Aims In pancreatic neuroendocrine neoplasms (pNEN) prognosis and therapeutic choices are mostly influenced by histological grading (G), based on Ki67 proliferative index: a correct pre-surgical evaluation is fundamental. The aim of the study is to evaluate the agreement of Ki67 and the respective grading, between EUS-guided sampling (FNA/FNB) and surgical specimen, in patients with pNEN.

Aims Macroscopic on-site evaluation (MOSE) looks promising for estimating the adequacy of EUS-FNB samples, in order to reduce the number of passes needed to achieve a diagnosis.

Merkel cell carcinoma is a rare (∼ 2000 cases/year in the USA) but aggressive neuroendocrine neoplasm of the skin. In 2008, the Merkel cell polyomavirus (MCPyV) was found to be clonally integrated in approximately 80% of Merkel cell carcinomas. The remaining 20% have large numbers of UV-associated mutations. Importantly, both the UV-induced neoantigens in virus-negative Merkel cell carcinoma and the Merkel cell polyomavirus oncogenes that are required for virus-positive tumor growth are highly immunogenic. Indeed, antigen-specific T cells detected in patients are frequently "dysfunctional/exhausted," and the inhibitory ligand PD-L1 is often expressed by Merkel cell carcinoma cells. These data led to point our attention on the quantity and the quality of the immune response in Merkel cell carcinoma. Here, we found CD8+ lymphocytes are the only singly evaluated lymphocyte subclass that strongly influenced overall survival and disease-specific survival in Merkel cell carcinoma. In addition, we highlighted as Merkel cell polyomavirus is a strong prognostic factor and as it prompts a host immune response involving various lymphocyte subclasses (CD3, CD8, FoxP3, and PD-L1 positive) in MCC. For this reason, we proposed a novel eye-based "immunoscore" model, obtained by tumor infiltrating lymphocytes subtyping (CD3, CD8, FoxP3, and PD-L1) that could provide additional prognostic information in Merkel cell carcinoma.

In 2014 a 44 years-old lady underwent transabdominal US for mild crampy abdominal pain and loose stools (eventually attributed to IBS). A 1.5 cm lesion in the uncinate process of the pancreas was detected (confirmed by CT). Then she was referred for EUS-FNA. A 13 × 11 mm hypoechoic mass was detected. FNA was performed with a 25G needle (4 passes). Cytology revealed sheets and bundles of spindle cells and immunohistochemistry led to the diagnosis of pancreatic schwannoma (negative staining for desmin, actin, DOG-1 and CD117 and positive for S100) with a low proliferation index (ki67 < 1%). Schwannomas are rare mesenchymal tumors, their origin from the pancreas is exceedingly rare (only 70 cases in literature). Their diagnosis before surgery is difficult, only 17 patients underwent EUS-FNA and the diagnosis could be reached in only 9 cases. To our knowledge, follow-up has been proposed to only 1 patient but the lesion grew up after a 11-month follow-up. After collegial consultation follow-up was proposed to our patient, relying to abdominal US. Four years have passed: the lesion is unchanged and the patient is asymptomatic. We think that a wait-and-see strategy may be proposed for pancreatic schwannomas, provided they are small, stable in size, with a low proliferation index, well-defined margins and without the need for excessive radiation exposure.

The American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2013 guidelines for HER2 assessment have increased the number of HER2 equivocal breast carcinomas following in situ hybridization reflex testing, that is, HER2 “double equivocal” (equivocal protein expression and equivocal gene copy number). Forty-five double-equivocal carcinomas were subjected to Prosigna analysis. Twenty-seven cases were investigated for the expression of genes found to be differentially expressed between estrogen receptor (ER)-positive/HER2-positive (N=22) and ER-positive/HER2-negative (N=22) control cases. Twenty-nine of the 45 cases were also analyzed by targeted sequencing using a panel of 14 genes. We then explored the pathologic complete response rates in an independent series of double-equivocal carcinoma patients treated with trastuzumab-containing chemotherapy. All cases were ER-positive, with a mean Ki67 of 28%. Double-equivocal carcinomas were predominantly luminal B (76%); 9 cases (20%) were luminal A, and 2 cases (4%) HER2-enriched. The majority (73%) showed a high risk of recurrence by Prosigna, even when the carcinomas were small (<2 cm), node-negative/micrometastatic, and/or grade 2. Double-equivocal carcinomas showed TP53 (6/29, 20%), PIK3CA (3/29, 10%), HER2 (1/29, 3%), and MAP2K4 (1/29, 3%) mutations. Compared with grade-matched ER-positive/HER2-negative breast carcinomas from METABRIC, double-equivocal carcinomas harbored more frequently TP53 mutations and less frequently PIK3CA mutations (P<0.05). No significant differences were observed with grade-matched ER-positive/HER2-positive carcinomas. Lower pathologic complete response rates were observed in double-equivocal compared with HER2-positive patients (10% vs. 60%, P=0.009). Double-equivocal carcinomas are preferentially luminal B and show a high risk of recurrence. A subset of these tumors can be labeled as HER2-enriched by transcriptomic analysis. HER2 mutations can be identified in HER2 double-equivocal cases.

Abstract BACKGROUND AND RATIONALE: Immunohistochemistry (IHC) and in situ hybridization (ISH) represent the cornerstone of HER2 evaluation in breast cancer (BC). We have demonstrated that up to 13% of BCs with equivocal HER2 expression (score 2+ in IHC) harbor an equivocal HER2 gene status (HER2/CEP17 <2 and mean HER2 copy number between 4 and 6), leading to the definition of the double-equivocal category. When we assessed HER2 gene levels in these cases by a PCR-based method we observed copy gain in 25%. When we tested HER2 protein levels by an in situ quantitative assay, HER2 levels ranged from those of score 0/ISH- to those observed in score 2+/ISH+ BCs. These data suggest that, rather than exploring alternative methods to assess HER2 status, a complementary functional approach may be beneficial. Our aim was to stratify double-equivocal BCs using global transcriptomics. METHODS & RESULTS: We retrieved a series of 27 formalin fixed paraffin embedded double-equivocal BCs and two control groups matched for oestrogen receptor and histological grade: 22 HER2- (IHC score 0/ISH-) and 22 HER2+ (IHC score 3+/ISH+) BCs. RNA was extracted following microdissection to enrich for tumor cell content >80% and subjected to whole-Genome DASL (cDNA-mediated Annealing, Selection, extension and Ligation; Illumina). We first identified genes with differential expression in HER2+ versus HER2- BCs based on T-test significance (p<0.01) and on mean gene expression variations higher than +/- 2-fold. To best characterize the signature we performed a cluster analysis with the GEDAS software using the "Fuzzy Self-organizing Maps" algorithm and the cosenic distance to generate the clusters. The classifier (24 genes) tested on double-equivocal cases led to a separation in "HER2+ like" and "HER2- like" BCs. More in details, three main clusters emerged, showing a significantly different distribution of cases with distinct HER2 status (p<0.0001, Chi square). Cluster A included all HER2+ and 5 double-equivocal cases and was associated to high expression of genes that pertain to the HER2 amplicon. Cluster B, composed of 9 HER2- and 12 double-equivocal cases, showed low expression of HER2 and HER2 amplicon-related genes and high levels of expression of TPRG1, NOVA1, AGTR1, SEZ6L, MAPT, GSTM1, SORCS1, DSCR6, NPY1R genes. Cluster C, formed by 13 HER2- and 10 double-equivocal cases, showed low expression of HER2 and HER2 amplicon-related genes but a non-homogeneous expression of the other genes of the classifier. The expression of best performing genes of the classifier (TPRG1, NOVA1, AGTR1) was investigated in The Cancer Genome Atlas (TCGA) dataset of breast cancer (Breast Invasive Carcinoma, TCGA provisional from cbioportal.org) and a striking mutually exclusive pattern with HER2 expression was observed. CONCLUSION: By resolving HER2 double-equivocal BCs into HER2 likely-positive or likely-negative, this approach may pave the way to an informed therapeutic decision in this controversial category of patients. Citation Format: Marchio C, Trisolini E, Maletta F, Annaratone L, Scalzo MS, Mascali D, Verdun di Cantogno L, Medico E, Sapino A. Stratification of breast carcinomas with double-equivocal HER2 status. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-05-04.

Background: MTOR inhibitors are approved for the treatment of advanced neuroendocrine tumors. However, scarce data are available on clinical or pathological predictors of response to these agents. The aim of the study is to test in a pilot series the presence of clinical and pathological predictors of response to mTOR inhibitors. Patients and methods: Clinical and pathological characteristics (sex, age, location of the primary tumor, tumor grade and Ki-67 index) and the expression of phosphorylated forms of mTOR and p70S6K were correlated with response to therapy in 20 neuroendocrine tumor patients treated with everolimus. Results: Seven patients had partial response (PR), 10 were stable (SD) and three progressed (PD) under everolimus treatment. Pancreatic location and higher mean Ki-67 index were significantly associated with a better response (p = 0.03 and p = 0.04, respectively). Phospho-mTOR and p-S6K levels were significantly correlated each other (p < 0.0001). No differences in p-mTOR and p-p70S6K expression levels were observed in tumors segregated according to tumor site. In paired matched samples, the expression of p-mTOR was increased in 5 out of 9 metastatic tissues, compared to the corresponding primary tumors. p-mTOR and p-p70S6K were not significantly correlated with response to treatment. However, all PD and a minority of PR/SD cases had high expression levels of p-mTOR. Conclusions: Some specific tumor characteristics seem to be associated with response to mTOR inhibitors and should be validated in larger series.

To identify markers of non-response to neoadjuvant chemotherapy (NAC) that could be used in the adjuvant setting. Sixteen pathologists of the European Working Group for Breast Screening Pathology reviewed the core biopsies of breast cancers treated with NAC and recorded the clinico-pathological findings (histological type and grade; estrogen, progesterone receptors, and HER2 status; Ki67; mitotic count; tumor-infiltrating lymphocytes; necrosis) and data regarding the pathological response in corresponding surgical resection specimens. Analyses were carried out in a cohort of 490 cases by comparing the groups of patients showing pathological complete response (pCR) and partial response (pPR) with the group of non-responders (pathological non-response: pNR). Among other parameters, the lobular histotype and the absence of inflammation were significantly more common in pNR ( p < 0.001). By ROC curve analyses, cut-off values of 9 mitosis/2 mm 2 and 18 % of Ki67-positive cells best discriminated the pNR and pCR + pPR categories ( p = 0.018 and < 0.001, respectively). By multivariable analysis, only the cut-off value of 9 mitosis discriminated the different response categories ( p = 0.036) in the entire cohort. In the Luminal B/HER2− subgroup, a mitotic count <9, although not statistically significant, showed an OR of 2.7 of pNR. A lobular histotype and the absence of inflammation were independent predictors of pNR ( p = 0.024 and <0.001, respectively). Classical morphological parameters, such as lobular histotype and inflammation, confirmed their predictive value in response to NAC, particularly in the Luminal B/HER2− subgroup, which is a challenging breast cancer subtype from a therapeutic point of view. Mitotic count could represent an additional marker but has a poor positive predictive value.

OBJECTIVE:Lymphoid proliferations of the salivary glands can be either reactive or malignant. Diagnosis based solely on fine needle aspiration (FNA) cytology may be troublesome in view of the difficulty in distinguishing low-grade B-cell and mucosa-associated lymphoid tissue (MALT) lymphomas from reactive lymphoid proliferations. We report our experience with FNA cytology combined with flow cytometry (FC) immunophenotyping for the diagnosis of lymphoproliferative processes affecting the salivary glands.METHODS:Sixty-one FNA specimens, obtained from salivary glands over a 10-year period, were analysed by cytology and FC. The results were correlated with histological follow-up if available.RESULTS:A diagnosis of lymphoma was given in 37 of 61 (61%) specimens; 22 of 61 (36%) specimens were considered as benign/reactive or non-lymphomatous processes; two of 61 (3%) specimens were considered as suspicious for lymphoma on cytological analysis and negative on FC. Histological control was available in 23 malignant, four non-lymphomatous and one cytologically suspicious case. Data obtained by the combination of cytology and FC were confirmed in all but one case: the case suspicious on cytology received a histological diagnosis of carcinoma. Four of seven cases with small populations of clonal cells (less than 15%) were histologically confirmed as lymphoma, whereas two remain under surveillance and one was reactive. Correlation with histological data showed a sensitivity of 100% and a specificity of 83% for the combination of cytology and FC.CONCLUSIONS:FC is fundamental for the diagnosis of lymphoproliferative lesions of the salivary glands. It may solve cytologically suspicious cases and detect the presence of neoplastic B or T cells. This combined approach reduces the time to therapy and may prevent unnecessary surgical biopsies.

A 64-year-old woman presented with longstanding dysphagia to solids and a recent onset of nocturnal cough and change in her voice. The patient described restriction to the passage of solids at the level of the neck, but denied odynophagia or weight loss. She had a background of Behcet’s disease with previous colonic and oropharyngeal involvement. She had also been treated for many years with esophageal dilations for an upper esophageal web. Her symptoms of dysphagia improved temporarily after each dilation; however, typically there was recurrence within 2 weeks of treatment. Video fluoroscopy was performed, which demonstrated a small web in the region of the pharyngo-esophageal junction, above which there was pharyngeal dilatation. At least one episode of aspiration occurred during this procedure. After informed consent had been obtained from the patient, repeat esophagogastroduodenoscopy was undertaken using a 5-mm pediatric endoscope (GIF-180; Olympus, Ontario, Canada). Multiple fibrotic-looking rings were noted throughout the hypopharynx (●" Fig.1a) and pharynx (●" Fig.1b,c). There was no ulceration or erythema present. A small diverticulum was seen just above the larynx (●" Fig.1d). The pediatric endoscope was passed into the esophagus, stomach, and duodenum with no other abnormalities being detected. The patient was referred to an ear, nose, and throat (ENT) surgeon for further management. ENT examination confirmed the presence of several fibrotic rings affecting the pharynx and hypopharynx with considerable narrowing toward the glottic opening. It was felt that the degree of restriction at the oropharynx was sufficient to account for the patient’s symptoms. The etiology of the rings remained unclear. Because of her persistent symptoms, it was recommended to the patient that she undergo excision of the rings during direct laryngoscopy using a laser. Histopathology of the specimens confirmed squamous mucosa with submucosal fibrosis and mild chronic inflammatory changes. At a follow-up visit after 2 months, the patient reported considerable improvement in her symptoms. Behcet’s disease is a type of systemic vasculitis that may affect small, medium, and large vessels (arteries or veins). The diagnosis of Behcet’s disease is a clinical one and requires the presence of recurrent mouth aphthous ulcers (at least three times per year), and two of the following: recurrent genital aphthous ulcers, eye lesions, skin lesions, or a positive pathergy test [1]. In the oropharynx, pharyngeal stenosis has been reported in addition to ulceration and it has been postulated that this is secondary to myositis [2]. Although there are reported cases of cicatricial pharyngeal stenosis in patients with Behcet’s syndrome and previous oropharyngeal surgery, this is the first reported case of pharyngeal webs associated with this syndrome. Established associations with pharyngeal webs include iron deficiency anemia, pernicious anemia, rheumatoid arthritis, carcinoma, epidermolysis bullosa, and pemphigoid [3]. Webs in the pharynx are a rare cause of dysphagia that should be considered in patients with persistent dysphagia once esophageal lesions and motility disorders have been excluded. Behcet’s disease may be associated with pharyngeal webs.